ISO 18337:2015

Surface chemical analysis — Surface characterization — Measurement of the lateral resolution of a confocal fluorescence microscope

ISO18337:2015 describes a method for determining the lateral resolution of a confocal fluorescence microscope (CFM) by imaging an object with a size much smaller than the expected resolution.

Analyse chimique des surfaces — Caractérisation des surfaces — Mesurage de la résolution latérale d'un microscope confocal à fluorescence

General Information

• Status: Published
• Publication Date: 18-Jun-2015
• ICS: 71.040.40 - Chemical analysis
• Technical Committee: ISO/TC 201 - Surface chemical analysis
• Drafting Committee: ISO/TC 201/WG 5 - Optical interface analysis
• Current Stage: 9093 - International Standard confirmed
• Completion Date: 30-Jun-2022

Standards Content (Sample)

INTERNATIONAL ISO
STANDARD 18337
First edition
2015-06-15
Surface chemical analysis — Surface
characterization — Measurement of
the lateral resolution of a confocal
fluorescence microscope
Analyse chimique des surfaces — Caractérisation des surfaces
— Mesurage de la résolution latérale d’un microscope confocal à
fluorescence
Reference number
ISO 18337:2015(E)
©
ISO 2015

---------------------- Page: 1 ----------------------
ISO 18337:2015(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2015, Published in Switzerland
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of
the requester.
ISO copyright office
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. [phone]
Fax [phone]
[e-mail]
www.iso.org
ii © ISO 2015 – All rights reserved

---------------------- Page: 2 ----------------------
ISO 18337:2015(E)

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Terms and definitions . 1
3 Symbols and abbreviated terms . 1
4 General . 1
4.1 Background information . 1
4.2 Types of CFM operation . 2
4.2.1 General. 2
4.2.2 Stage scanning CFM . 2
4.2.3 Laser scanning CFM . 2
4.2.4 Spinning disk CFM . 2
4.3 Parameters that affect the lateral resolution of a CFM . 3
4.3.1 General. 3
4.3.2 Objective lens . 3
4.3.3 Detection pinhole size and focal length of the tube lens . 3
4.3.4 Collimation and purity of the laser illumination beam . 3
4.3.5 Polarization of the laser illumination . 3
4.3.6 Excitation and emission wavelengths . 3
4.3.7 Image contrast . 3
5 Measuring the lateral resolution by imaging a small object . 3
5.1 Background information . 3
5.2 Selection of the sample and sample requirements . 4
5.3 Setting the parameters prior to operating the instrument . 4
5.4 Data collection and analysis . 5
5.4.1 Selecting a proper spot . 5
5.4.2 Extracting a line profile using band-average process . 5
5.5 Recording the data . 6
Annex A (informative) Sample preparation and example of data and analysis .7
Bibliography .10
© ISO 2015 – All rights reserved iii

---------------------- Page: 3 ----------------------
ISO 18337:2015(E)

Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of any
patent rights identified during the development of the document will be in the Introduction and/or on
the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the meaning of ISO specific terms and expressions related to conformity
assessment, as well as information about ISO’s adherence to the WTO principles in the Technical Barriers
to Trade (TBT) see the following URL: Foreword - Supplementary information
The committee responsible for this document is ISO/TC 201 Surface chemical analysis.
iv © ISO 2015 – All rights reserved

---------------------- Page: 4 ----------------------
ISO 18337:2015(E)

Introduction
Confocal fluorescence microscopes (CFMs) are laser scanning confocal microscopes (LSCMs) operated
in a fluorescence imaging mode so as to obtain a fluorescence image of a sample. Fluorescence is the
light emitted by a molecule or solid lattice during relaxation after undergoing photon absorption and
electronic excitation. The fluorescence wavelength, intensity and spectral shape are specific to the
electronic structure of the material; therefore, fluorescence spectroscopy and imaging techniques are
useful for chemical characterization and analysis. Among the optical imaging and spectroscopy tools,
CFM yields a high spatial resolution that is advantageous for analysing nanomaterials and thin films.
The spatial resolution is one of the most important performance factors for a CFM.
The spatial resolution of a technique refers to the maximum resolvability of two adjacent objects.
This value is often characterized in different ways by the manufacturers. The spatial resolution of a
CFM is characterized by both the lateral and axial resolution, which have different values and are not
necessarily dependent on one another. In this International Standard, one convenient and effective
method for measuring the lateral resolution of a CFM is presented. This method is suitable for use by
non-expert operators.
© ISO 2015 – All rights reserved v

---------------------- Page: 5 ----------------------
INTERNATIONAL STANDARD ISO 18337:2015(E)
Surface chemical analysis — Surface characterization
— Measurement of the lateral resolution of a confocal
fluorescence microscope
1 Scope
This International Standard describes a method for determining the lateral resolution of a confocal
fluorescence microscope (CFM) by imaging an object with a size much smaller than the expected
resolution.
2 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
2.1
point spread function
response of an imaging system to a point source or point object
2.2
lateral resolution
distance measured either in the plane of the sample surface or in a plane at right angles to the axis of the
image forming optics over which changes in composition can be separately established with confidence
[1]
Note 1 to entry: See Reference
.
3 Symbols and abbreviated terms
OL objective lens
APD avalanche photodiode
FWHM full width at half-maximum
CFM confocal fluorescence microscope
NA numerical aperture
PSF point spread function
QD quantum dot
PMT photomultiplier tube
4 General
4.1 Background information
Laser scanning confocal microscopes (LSCMs) scan a tightly focused laser beam over a sample and
record the optical intensity at each pixel to form a two-dimensional image. LSCMs have a pinhole in front
of the photo detector or a spectrometer input slit at the conjugate focal plane of the laser focus at the
sample. Light originating from the non-focal plane is largely prevented from reaching the photo detector.
Confocal configurations significantly improve the contrast in an image, and the spatial resolution may
[2]
be increased
.
© ISO 2015 – All rights reserved 1

---------------------- Page: 6 ----------------------
ISO 18337:2015(E)

CFM is one of the most widely used LSCM operation modes because it provides a fluorescence image
or spectrum. In the fluorescence mode, the incident laser light is blocked by a long pass filter, and only
the Stokes-shifted fluorescence light is detected by the photo detector or spectrometer. An image is
formed, depending on the fluorescence wavelength, and the image displays good contrast compared to
images obtained through other optical imaging techniques. Imaging multiple colours in one image is
also possible.
The spatial resolution is one of the most important features characterizing the performance of an LSCM
or CFM. The lateral resolution and axial resolution must be determined separately and must be treated
independently. The lateral resolution is important especially when a CFM is used for the imaging and
chemical analysis of thin films or nanoscale objects, in which the axial dimension is significantly less
than the typical value of the axial resolution of a CFM.
The spatial resolutions of instruments tend to be characterized in different ways by different
manufacturers. This work provides one convenient and effective method for measuring the lateral
spatial resolution of a CFM instrument so as to be suitable for use by a non-expert operator. The terms
[3]
and analysis procedure described here are according to ISO 18516
.
4.2 Types of CFM operation
4.2.1 General
Below, we describe different modes of CFM operation according to the laser focus scanning technique
with respect to the sample. This International Standard principally treats stage scanning-type and laser
scanning-type CFMs.
4.2.2 Stage scanning CFM
Stage scanning CFMs are characterized by a moving stage that implements a scanning function while
the laser focus remains unmoved with respect to the microscope frame. Because the optics involved in
the CFM are stationary, the beam path is simple and nearly maintenance-free. Aberrations or drift in the
optical alignment are minimized. The scan area size is limited only by the mechanical movement of the
sample scanning stage, and allows for large-area scans.
Scanning speeds in these CFMs are relatively slow, and the sample may be affected by any rapid
movements of a scanning stage and, therefore, may not be suitable for imaging delicate cells.
4.2.3 Laser scanning CFM
Off-axis beam scanning techniques can incur aberrations that degrade the image resolution. A fast
oscillating mirror set may be used to scan a beam across a sample more rapidly than is possible in stage
scanning-type CFM. The scanning rate can sometimes reach values of a few kHz line scan speed. In this
approach, the sample does not move, thereby preserving the condition of the sample.
Because the optical ray is off-axis during laser focus scanning, some aberrations may be introduced into
the image. The need for a scanning head makes this technique more complicated than techniques based
on stage scanning. The size of a scan area is limited because an objective will admit off-axis laser light
within only limited angle. The size of a scanning area also depends on the magnification of the objective.
The use of low magnification objectives (resulting in a large scan area) which at the same time have a
high NA (which results in a good collection efficiency and an improved resolution) is preferential in this
case.
4.2.4 Spinning disk CFM
High-speed spinning disks involve multiple laser foci and conjugated detector pinholes.
2 © ISO 2015 – All rights reserved

---------------------- Page: 7 ----------------------
ISO 18337:2015(E)

4.3 Parameters that affect the lateral resolution of a CFM
4.3.1 General
Lateral resolution assessments are made by measuring the lateral size of a PSF on a CFM. The fundamental
limit in the spatial resolution of a light microscope is given by:
2
Abbe Resolution = λ/2NA, Abbe Resolution = 2λ/NA
x,y z
Comprehensive reviews of the relevant parameters and an experimental protocol for determining the
[2] [5] [6] [7]
size of a PSF are available elsewhere , , , .
4.3.2 Objective lens
The objective lens (OL) directly affects the lateral resolution of a CFM image because the size of the laser
focus and the collection volume are determined by the NA of the OL.
4.3.3 Detection pinhole size and focal length of the tube lens
The confocal pinhole in front of a photo detector eliminates light originating outside of the focal volume
in the sample. Smaller pinholes increase the resolution; however the amount of light that contributes to
the image is also reduced, which can reduce the contrast in the image. Therefore the optimum size of
the detection pinhole for a particular sample and application must be selected with the discretion of the
operator. The imaged size of the detection pinhole on the sample plane by the tube lens and the OL is
more important than the actual size of the detection pinhole. Therefore the magnification of the OL and
the tube lens should also be specified.
4.3.4 Collimation and purity of the laser illumination beam
The expected performance of an OL can be achieved provided that the illumination beam is collimated
and fills the back aperture of the OL.
4.3.5 Polarization of the laser illumination
The polarization of the input laser affects the shape and size of a PSF. Linearly polarized illumination
[8]
tends to elongate the PSF along the direction of polarization .
4.3.6 Excitation and emission wavelengths
The wavelength of the light used in CFM has the direct effect on the size of PSF as described by Abbe
resolution in 4.3.1. Furthermore, in the fluorescence imaging, the emission wavelength is different from
[2]
(longer than) the excitation wavelength and this can lower the resolution .
4.3.7 Image contras
...